Phospholipase

ABSTRACT

The present invention is related to a method for producing a phospholipase by processing an expressed fungal peptide and to certain specified phospholipases. Furthermore the invention provides a method for producing cheese with a phospholipase.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisionalapplication No. 60/467,865, filed May 5, 2003, and 60/496,158, filed,Aug. 19, 2003, and priority of Danish Patent Application Nos. PA 200300634, filed Apr. 28, 2003, and PA 2003 001163, filed Aug. 14, 2003 thecontents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of hydrolyzing aphospholipid, a method of producing a phospholipase, a method of makingcheese, and to a phospholipase.

BACKGROUND OF THE INVENTION

[0003] Soragni, E., et al. (2001) EMBO J. 20: 5079-5090 discloses aphospholipase (TbSP1) from Tuber borchii and the nucleotide sequence ofa cDNA of a gene encoding it. The following peptide sequence arepublished in the indicated sources, derived from the indicated sourceorganism:

[0004] COGEME Phytopathogenic Fungi and Oomycete EST Database,Unisequence ID: VD0100C34, Verticillium dahliae.

[0005] NCBI Protein database, gi: 18307435, Neurospora crassa

[0006] NCBI Protein database, gi: 16519372, Helicosporum sp. HN1

[0007] WO 0056762, SEQ ID NO: 5954, Aspergillus oryzae

[0008] TREMBL Protein database, EAA28927, Neurospora crassa U.S. Pat.No. 6,399,121 discloses the use of phospholipase in cheese making.

SUMMARY OF THE INVENTION

[0009] The inventors have analyzed known sequence data for fungal GroupXIII phospholipases A2, and they have identified additional sequences,either from published sequence data or by screening for relevantsequences from natural sources. By expressing genes encoding fungalGroup XIII phospholipases A2 in a suitable host organism they found thatthe expressed sequences consist of a core peptide coupled to a peptidesequence at the N- or C-terminal side, or both, and that expression ofthe gene in a suitable host organism can lead to cleavage of theexpressed peptide to obtain the core peptide without any peptideextension at the N- or C-terminal. They further found that the corepeptide without any peptide extension(s) has a significantly higherphospholipase activity than the core peptide linked to the peptideextension(s). Finally, they found that the core peptide discovered bythis method is similar in length and sequence to a known mature peptidefrom Helicosporium sp. (Wakatsuki, S. et al. (2001) Biochim. Biophys.Acta 1522: 74-81) of unknown function, and to bacterial Group XIIIphospholipases A2, which lack peptide extensions other than secretionsignals (Sugiyama, M. et. al. (2002) J. Biol. Chem. 277:20051-20058).

[0010] The inventors additionally found that phospholipase sharing theactive site sequence similarity and cysteine residue conservation offungal Group XIII phospholipase A2 is useful in cheese making.

[0011] Additionally, the inventors discovered and isolated a geneencoding a novel phospholipase from Fusarium venenatum A3/5, which wasoriginally deposited as Fusarium graminearum ATCC 20334 and recentlyreclassified as Fusarium venenatum by Yoder and Christianson, 1998,Fungal Genetics and Biology 23: 62-80; and O'Donnell et al., 1998,Fungal Genetics and Biology 23: 57-67. The phospholipase belongs to thefungal/bacterial group XIII PLA2 as defined by Soragni et al., The EMBOJournal, 20 (2001), 5079-5090. The inventors also cloned the novelphospholipase encoding gene into an E. coli strain, and used the clonedgene to make a construct for expressing the Fusarium phospholipase genein Aspergillus oryzae. The inventors transformed Aspergillus oryzae withthis construct, and isolated the phospholipase from transformedAspergillus cells.

[0012] Accordingly, the invention provides a method of producing aphospholipase which comprises processing an expressed fungal peptide soas to cleave off a peptide from the C-terminal end and/or a peptide fromthe N-terminal end to obtain a core peptide, wherein the core peptidecomprises:

[0013] a) the amino acid sequence given by amino acids 146-153 of SEQ IDNO: 1, amino acids 87-94 of SEQ ID NO: 3, or amino acids 79-86 of SEQ IDNO: 12; or a sequence identical to any of these amino acid sequencesexcept for the substitution of a single amino acid with another aminoacid; and

[0014] b) at least two cysteine residues located on the N-terminal sideof the sequence given in a); and

[0015] c) at least two cysteine residues located on the C-terminal sideof the sequence given in a).

[0016] The invention also provides a method for hydrolyzing aphospholipid with a phospholipase of the invention. Furthermore theinvention provides a method for producing cheese by contacting cheesemilk or a fraction of cheese milk with a phospholipase and producingcheese from the cheese milk.

[0017] Finally, the invention provides phospholipase which is apolypeptide having an amino acid sequence which is at least 80%identical with certain specified sequences.

BRIEF DESCRIPTION OF DRAWINGS

[0018]FIG. 1 shows an alignment of amino acid sequences of fungal groupXIII phospholipases A2, showing processing sites (|) where known. Theactive site consensus is underlined. Conserved cysteine residues areindicated with | under the consensus. Alignment was made with the AlignXprogram of the Vector NTI program suite v8. The algorithm used isClustalW with the blosum62mt2 matrix and AlignX default settings.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Expressed Peptide

[0020] The invention uses an expressed fungal peptide belonging to agroup defined by the active site sequence similarity and cysteineresidue conservation used in the definition of the group“fungal/bacterial group XIII phospholipase A2” given by Soragni, E., etal. (2001) EMBO J. 20: 5079-5090. The peptide is fungal, e.g. derivedfrom Tuber, Verticillium, Neurospora, Helicosporum, or Aspergillus,particularly T. borchii, T. albidum, V. dahliae, V. tenerum, N. crassa,Helicosporium sp. HN1 or A. oryzae.

[0021] The peptide may have phospholipase activity, e.g. phospholipase Aactivity, such as phospholipase A1 and/or phospholipase A2 activity.

[0022] Some particular examples are known peptides having amino acidsequences listed in the sequence listing as follows. The sourceorganisms and literature references are also indicated:

[0023] SEQ ID NO:1. Tuber borchii. Soragni, E., et al. (2001) EMBO J.20: 5079-5090

[0024] SEQ ID NO: 3. Verticillium dahliae. COGEME Phytopathogenic Fungiand Oomycete EST Database, Unisequence ID: VD0100C34.

[0025] SEQ ID NO: 4. Neurospora crassa. NCBI Protein database, gi:18307435.

[0026] SEQ ID NO: 5. Helicosporum sp. HN1. NCBI Protein database, gi:16519372.

[0027] SEQ ID NO: 7. Aspergillus oryzae. WO 0056762, SEQ ID NO: 5954.

[0028] SEQ ID NO 8. Neurospora crassa. TREMBL Protein database, EAA28927

[0029] Further, the following fungal phospholipases having the indicatedsequences were isolated by the inventors from natural sources purchasedfrom public collections or collected in the indicated country and year:

[0030] SEQ ID NO: 10. Tuber albidum. Purchased from Centraalbureau voorSchimmelcultures, Utrecht, The Netherlands, isolate CBS272.72

[0031] SEQ ID NO: 12. Verticillium tenerum. Ireland, 1996

[0032] The inventors inserted the gene from T. albidum (SEQ ID NO: 9)into E. coli and deposited the clone under the terms of the BudapestTreaty on the 12 Feb. 2003. The deposit was made at the Deutsce Sammlungvon Mikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1b, D-38124Braunschweig, Germany, and was accorded deposit number DSM 15441.

[0033] In one embodiment the invention provides a phospholipase which isa polypeptide having an amino acid sequence which is at least 80%, suchas at least 85%, preferably 90%, more preferably at least 95%, identicalwith amino acids 91-210 in SEQ ID NO: 10 (T. albidum), amino acids92-211 in SEQ ID NO: 1 (T. borchil), amino acids 30-137 in SEQ ID NO: 12(V. tenerum), amino acids 38-145 in SEQ ID NO: 3 (V. dahliae), aminoacids 44-151 in SEQ ID NO: 4 (N. crassa), amino acids 37-157 in SEQ IDNO: 7 (A. oryzae), or amino acids 58-168 in SEQ ID NO: 8 (N. crassa).

[0034] Peptide Processing

[0035] By analyzing the phospholipase sequences in the sequence listing,the inventors found that each expressed amino acid sequence consists ofa signal peptide, a core peptide, and additionally a peptide sequencewith unknown function attached to the C- or N-terminal, or both, of thecore peptide.

[0036] Core Peptide

[0037] The core peptides are characterized by the same active sitesequence similarity and cysteine residue conservation observed bySoragni, E., et al. (2001) EMBO J. 20: 5079-5090 for thefungal/bacterial group XIII phospholipase A2.

[0038] In a preferred embodiment of the invention the core peptidescomprises: a) the sequence given by amino acids 146-153 of SEQ ID NO: 1,amino acids 87-94 of SEQ ID NO: 3, or amino acids 79-86 of SEQ ID NO:12; or a sequence identical to any of these amino acid sequences exceptfor the substitution of a single amino acid with another amino acid; andb) two cysteine residues located on the N-terminal side of the sequencegiven in a); and c) two cysteine residues located on the C-terminal sideof the sequence given in a).

[0039] One of the cysteine residues located on the N-terminal side ofthe sequence given in a), may e.g. be separated from the sequence givenin a) by 0-5 amino acids, such as 0-3 amino acids, preferably 0-2 aminoacids, and even more preferably 1 amino acid. Another of the cysteineresidues located on the N-terminal side of the sequence given in a) maye.g. be separated from the sequence given in a) by 14-20 amino acids,such as 15-19 amino acids, preferably 16-18 amino acids, and even morepreferably 17 amino acids.

[0040] One of the cysteine residues located on the C-terminal side ofthe sequence given in a), may e.g. be separated from the sequence givenin a) by 22-29 amino acids, such as 23-28 amino acids, preferably 24-27amino acids, and even more preferably 25-26 amino acids. Another of thecysteine residues located on the C-terminal side of the sequence givenin a) may e.g. be separated from the sequence given in a) by 27-49 aminoacids, such as 29-46 amino acids, preferably 30-43 amino acids, evenmore preferably 32-42 amino acids, and most preferably 35-40 aminoacids.

[0041] In a preferred embodiment the core peptide comprises fourcysteine residues aligning with the cysteine residues of SEQ ID NO:1with amino acid numbers 128, 144, 180, and 194, respectively, when thecomplete expressed phospholipase sequence is aligned simultaneously withthe sequences given in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12.

[0042] According to the invention, the expressed polypeptide is cleavedso as to separate the core peptide from the attached peptide(s). Thecleavage may be done in vivo by expressing it in a suitable filamentousfungal host or in vitro, e.g. by a treatment with a suitable proteasesuch as e.g. Kex2.

[0043] The cleavage points may be found within 11 amino acids of asequence which is FG or within 10 amino acids of a sequence which is aKex2 site. Kex2 sites are e.g. RR, KR, KK or RK. In one embodiment thecore peptide has a length of 100-150 amino acids, such as 110-140 aminoacids, 115-133 amino acids, 118-129 amino acids, or 118-126 amino acids.

[0044] In one embodiment of the invention the expressed phospholipase iscleaved within 0-18 amino acids, such as 3-16 amino acids, preferably5-14 amino acids on the N-terminal side of the sequence aligning withamino acids 97-101 of SEQ ID NO: 1, when the complete expressedphospholipase sequence is aligned simultaneously with the sequencesgiven in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12.

[0045] In a preferred embodiment the expressed phospholipase is cleavedwithin 0-11 amino acids, such as 0-9 amino acids, preferably 0-7 aminoacids, on the C-terminal side of the sequence aligning with amino acids204-209 of SEQ ID NO: 1, when the complete expressed phospholipasesequence is aligned simultaneously with the sequences given in SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 10, and SEQ ID NO: 12.

[0046] In a preferred embodiment the processed phospholipase has aspecific phospholipase activity, which is higher than the activity ofthe expressed peptide before processing, e.g. in one embodiment thespecific phospholipase activity is at least 2 times, more preferably atleast 5 times, most preferably at least 10 times the specificphospholipase activity of the expressed peptide before processing. Inone embodiment of the invention the expressed peptide does not havemeasurable phospholipase activity before processing.

[0047] Phospholipase activity may e.g. be measured in the LEU assay byhydrolyzing soy lecithin (L-alfa-phosphotidyl-choline) at pH 8.0 and 40°C. for 2 minutes. Phospholipase activity is expressed as the rate oftitrant consumption (0.1 M NaOH) necessary for keeping constant pH,relative to a standard.

[0048] Expression in Filamentous Fungal Host Cell

[0049] The filamentous fungal host cell may e.g. be a cell ofAcremonium, Aspergillus, Fusarium, Humicola, Myceliophthora, Neurospora,Penicillium, Rhizomucor, Thermomyces, Thielavia, Tolypocladium, orTrichoderma, particularly A. awamori, A. foetidus, A. japonicus, A.nidulans, A. niger, A. oryzae. F. bactridioides, F. cerealis, F.crookwellense, F. culmorum, F. graminearum, F. graminum, F.heterosporum, F. negundi, F. oxysporum, F. reticulatum, F. roseum, F.sambucinum, F. sarcochroum, F. sporotrichioides, F. sulphureum, F.torulosum, F. trichothecioides, F. venenatum, H. insolens, M.thermophila, N. crassa, P. purpurogenum, R. miehei, Thermomyceslanuginosus, Thielavia terrestris, Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, orTrichodenma viride.

[0050] In a preferred embodiment the host organism is a strain ofAspergillus, Fusarium, or Trichoderma, particularly A. niger, A. oryzae,F. venenatum, F. sambucinum or F. cerealis

[0051] The transformation, cultivation, expression, recovery may beperformed by conventional methods, e.g. by the general methods describedin EP 238023, EP 305216, WO 9600787, EP 244234 or T. Christensen et al.,BioTechnology, vol. 6, December 1988, 1419-22.

[0052] Phospholipase Polypeptide and DNA

[0053] In one embodiment, the present invention relates to polypeptideshaving phospholipase activity and where the polypeptides comprises,preferably consists of, an amino acid sequence which has a degree ofidentity to amino acids 29 to 149 of SEQ ID NO: 16 (i.e., the maturepolypeptide) of at least 80%, such as at least 85%, even more preferablyat least 90%, most preferably at least 95%, e.g. at least 96%, such asat least 97%, and even most preferably at least 98%, such as at least99%.

[0054] Preferably, the polypeptides comprise the amino acid sequence ofSEQ ID NO: 16; an allelic variant thereof; or a fragment thereof thathas phospholipase activity. In another preferred embodiment, thepolypeptide of the present invention comprises amino acids 29 to 149 ofSEQ ID NO: 16. In a further preferred embodiment, the polypeptideconsists of amino acids 29 to 149 of SEQ ID NO: 16.

[0055] The present invention also relates to a polynucleotidecomprising, preferably consisting of, a nucleotide sequence which has atleast 80% identity with nucleotides 133 to 495 of SEQ ID NO: 15.Preferably, the nucleotide sequence has at least 85% identity, such asat least 90% identity, more preferably at least 95% identity, such as atleast 96% identity, e.g. at least 97% identity, even more preferably atleast 98% identity, such as at least 99% with nucleotides 133 to 495 ofSEQ ID NO: 15. Preferably, the nucleotide sequence encodes a polypeptidehaving phospholipase activity.

[0056] The phospholipase may be derived from a strain of Fusarium,particularly F. venenatum, using probes designed on the basis of the DNAsequences in this specification. In one embodiment the phospholipase hasphospholipase A activity.

[0057] The phospholipase may be produced by transforming a suitable hostcell with a DNA sequence encoding the phospholipase, cultivating thetransformed organism under conditions permitting the production of theenzyme, and recovering the enzyme from the culture.

[0058] The host organism is preferably a eukaryotic cell, in particulara fungal cell, such as a yeast cell or a filamentous fungal cell, suchas a strain of Aspergillus, Fusarium, Trichoderma or Saccharomyces,particularly A. niger, A. oryzae, F. venenatum, F. sambucinum, F.cerealis or S. cerevisiae, e.g. a glucoamylase-producing strain of A.niger such as those described in U.S. Pat. No. 3,677,902 or a mutantthereof. The production of the phospholipase in such host organisms maybe done by the general methods described in EP 238,023 (Novo Nordisk),WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko).

[0059] The expression vector of the invention typically includes controlsequences functioning as a promoter, a translation initiation signal,and, optionally, a selectable marker, a transcription terminator, arepressor gene or various activator genes. The vector may be anautonomously replicating vector, or it may be integrated into the hostcell genome.

[0060] Sequence Alignment and Identity

[0061] Nucleotide sequences may be aligned with the AlignX applicationof the Vector NTI Program Suite 7.0 using the default settings, whichemploy a modified ClustalW algorithm (Thompson, J. D., Higgins, D. G.,and Gibson T. J. (1994) Nuc. Acid Res. 22: 4673-4680), the swgapdnarntscore matrix, a gap opening penalty of 15 and a gap extension penalty of6.66.

[0062] Amino acid sequences may be aligned with the AlignX applicationof the Vector NTI Program Suite v8 using default settings, which employa modified ClustalW algorithm (Thompson, J. D., Higgins, D. G., andGibson T. J., 1994), the blosum62mt2 score matrix, a gap opening penaltyof 10 and a gap extension penalty of 0.1.

[0063] In one embodiment of the invention alignments of sequences andcalculation of homology scores are done using the Lipman-Pearson Method(Lipman, D. J. and W. R. Pearson (1985) Rapid and sensitive proteinsimilarity searches. Science 227: 1435-1441) using a PAM250 residueweight table (Dayhoff, M. O., R. M. Schwartz, and B. C. Orcutt (1978) Amodel of evolutionary change in proteins. In Dayhoff, M. O. (ed.), Atlasof Protein Sequence and Structure. National Biomedical ResearchFoundation. Washington, D. C. Vol 5. Suppl. 3: pp. 345-358) and thedefault settings of the MegAlign program, v4.03, in the Lasergenesoftware package (DNASTAR Inc., 1228 South Park Street, Madison, Wis.53715). The default settings are a K-tuple of 2, gap penalty of 4, and agap length penalty of 12.

[0064] Phospholipid Hydrolysis

[0065] The invention may be used in the hydrolysis of any phospholipidsuch as a lecithin, a cephalin or an inositide.

[0066] The invention may be used in analogy with prior art processes byreplacing the phospholipase, e.g. in the production of baked products(WO 0032758, WO 9953769), mayonnaise (GB 1525929, U.S. Pat. No.4,034,124) or treatment of vegetable oil (U.S. Pat. No. 5,264,367).

[0067] Use of Phospholipase

[0068] The phospholipase of the invention can be used in variousindustrial application of phospholipases, e.g. as described below.

[0069] Use in Baking

[0070] The phospholipase of the invention can be used in the preparationof dough, bread and cakes, e.g. to improve the elasticity of the breador cake. Thus, the phospholipase can be used in a process for makingbread, comprising adding the phospholipase to the ingredients of adough, kneading the dough and baking the dough to make the bread. Thiscan be done in analogy with U.S. Pat. No. 4,567,056 or WO 99/53769.

[0071] Use in Detergent

[0072] The variant may be used as a detergent additive, e.g. at aconcentration (expressed as pure enzyme protein) of 0.001-10 (e.g.0.01-1) mg per gram of detergent or 0.001-100 (e.g. 0.01-10) mg perlitre of wash liquor.

[0073] The detergent composition of the invention may for example beformulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or be formulatedas a detergent composition for use in general household hard surfacecleaning operations. In a laundry detergent, the variant may beeffective for the removal of fatty stains, for whiteness maintenance andfor dingy cleanup. A laundry detergent composition may be formulated asdescribed in GB 2247025, WO 9901531 or WO 9903962.

[0074] The detergent composition of the invention may particularly beformulated for hand or machine dishwashing operations. e.g. as describedin GB 2,247,025 (Unilever) or WO 99/01531 (Procter & Gamble). In adishwashing composition, the variant may be effective for removal ofgreasy/oily stains, for prevention of the staining/discoloration of thedishware and plastic components of the dishwasher by highly coloredcomponents and the avoidance of lime soap deposits on the dishware.

[0075] Other Uses

[0076] The phospholipase of the invention can be used to improve thefilterability of an aqueous solution or slurry of carbohydrate origin bytreating it with the phospholipase. This is particularly applicable to asolution of slurry containing a starch hydrolyzate, especially a wheatstarch hydrolyzate, since this tends to be difficult to filter and togive cloudy filtrates. The treatment can be done in analogy with EP219,269 (CPC International).

[0077] Further, the phospholipase of the invention may be used forpartial hydrolysis of phospholipids, preferably lecithin, to obtainimproved phospholipid emulsifiers. This application is further describedin Ullmann's Encyclopedia of Industrial Chemistry (Publisher: VCHWeinheim (1996)), JP patent 2794574, and JP-B 6-087751.

[0078] Further, the phospholipase of the invention may be used in aprocess for the production of an animal feed which comprises mixing thephospholipase with feed substances and at least one phospholipid. Thiscan be done in analogy with EP 743 017.

[0079] Even further the phospholipase of the invention can be used in aprocess for reducing the content of phospholipid in an edible oil,comprising treating the oil with the phospholipase so as to hydrolyze amajor part of the phospholipid, and separating an aqueous phasecontaining the hydrolyzed phospholipid from the oil. This process isapplicable to the purification of any edible oil which containsphospholipid, e.g. vegetable oil such as soy bean oil, rape seed oil andsunflower oil. The phospholipase may e.g. be used in the processesdescribed in JP-A 2-153997 and U.S. Pat. No. 5,264,367.

[0080] Method for Producing Cheese

[0081] The phospholipase of the invention may be used for producingcheese in analogy with the process given in U.S. Pat. No. 6,399,121.

[0082] In a preferred embodiment of the invention cheese is produced bycontacting cheese milk or a fraction of cheese milk with a phospholipaseof the invention and producing cheese from the cheese milk.

[0083] In a further preferred embodiment cheese is produced bycontacting cheese milk or a fraction of cheese milk with aphospholipase, wherein the phospholipase comprises:

[0084] a) the sequence given by amino acids 146-153 of SEQ ID NO: 1,amino acids 87-94 of SEQ ID NO: 3, or amino acids 79-86 of SEQ ID NO:12; or a sequence identical to any of these amino acid sequences exceptfor the substitution of a single amino acid with another amino acid; and

[0085] b) two cysteine residues located on the N-terminal side of thesequence given in a); and

[0086] c) two cysteine residues located on the C-terminal side of thesequence given in a).

[0087] In the present context the term cheese milk is meant to cover anymilk based composition used for production of cheese. A fraction of thecheese milk may be any fraction of the cheese milk such as e.g. cream,skim milk, milk, butter milk, butter or milk fat.

[0088] In a preferred embodiment cheese milk or a fraction of cheesemilk is contacted with a phospholipase of the invention in an amountsufficient to decrease the oiling-off effect in cheese and/or toincrease cheese yield. The oiling-off effect is the tendency of thecheese to form free oil upon storage and/or melting.

[0089] In one aspect the invention relates to a process for producingcheese comprising treating a dairy composition with a phospholipase ofthe invention and producing cheese from the dairy composition.

[0090] Another aspect of the invention relates to a process forproducing cheese comprising treating a dairy composition withphospholipase and producing cheese from the dairy composition, whereinthe phospholipase is selected from the group of fungal/bacterial groupXIII PLA2 phospholipases. In a preferred embodiment of the invention thefungal/bacterial group XIII PLA2 is from a fungus, more preferably froma fungus belonging to the Ascomycetes.

[0091] A phospholipase belonging to the fungal/bacterial group XIII PLA2may be any phospholipase belonging to this group as defined by Soragniet al., The EMBO Journal, 20 (2001), 5079-5090, and may e.g. be from thespecies Tuber, e.g. T. borchii, Streptomyces, e.g. S. coelicor,Verticillium, e.g. V. dahliae, Aspergillus, e.g. A. oryzae, Neurospora,e.g. N. crassa, or Helicosporum.

[0092] A dairy composition according to the invention may be anycomposition comprising milk constituents. Milk constituents may be anyconstituent of milk such as milk fat, milk protein, casein, wheyprotein, and lactose. A milk fraction may be any fraction of milk suchas e.g. skim milk, butter milk, whey, cream, milk powder, whole milkpowder, skim milk powder. In a preferred embodiment of the invention thedairy composition comprises milk, skim milk, butter milk, whole milk,whey, cream, or any combination thereof. In a more preferred embodimentthe dairy composition consists of milk, such as skim milk, whole milk,cream, buttermilk, or any combination thereof.

[0093] The enzymatic treatment in the process of the invention may beconducted by dispersing the phospholipase into the dairy composition,and allowing the enzyme reaction to take place at an appropriateholding-time at an appropriate temperature. The treatment withphospholipase may be carried out at conditions chosen to suit theselected enzyme(s) according to principles well known in the art.

[0094] The enzymatic treatment may be conducted at any suitable pH, suchas e.g., in the range 2-10, such as, at a pH of 4-9 or 5-7. In oneembodiment the phospholipase treatment is conducted at 3-60° C., such asat 25-45° C. (e.g., for at least 5 minutes, such as, e.g., for at least10 minutes or at least 30 minutes, e.g., for 5-120 minutes). Thephospholipase is added in a suitable amount to produce the cheese havingthe desired properties. Preferably, the phospholipase is added in anamount effective to decrease the oiling-off effect in cheese and/or toincrease cheese yield. A suitable dosage of phospholipase will usuallybe in the range 0.001-0.5 mg enzyme protein per g milk fat, preferably0.01-0.3 mg enzyme protein per g milk fat, more preferably, 0.02-0.1 mgenzyme protein per g milk fat

[0095] The cheeses produced by the process of the present inventioncomprise all varieties of cheese, such as, e.g. Campesino, Chester,Danbo, Drabant, Herregard, Manchego, Provolone, Saint Paulin, Softcheese, Svecia, Taleggio, White cheese, including rennet-curd cheeseproduced by rennet-coagulation of the cheese curd; ripened cheeses suchas Cheddar, Colby, Edam, Muenster, Gruyere, Emmenthal, Camembert,Parmesan and Romano; blue cheese, such as Danish blue cheese; freshcheeses such as Feta; acid coagulated cheeses such as cream cheese,Neufchatel, Quarg, Cottage Cheese and Queso Blanco. In a preferredembodiment the invention relates to a process for producing pasta filatacheese, such as e.g. Mozzarella and Pizza cheese. Pasta filata, orstretched curd, cheeses are normally distinguished by a uniqueplasticizing and kneading treatment of the fresh curd in hot water,which imparts the finished cheese its characteristic fibrous structureand melting and stretching properties, cf. e.g. “Mozzarella and Pizzacheese” by Paul S. Kindstedt, Cheese: Chemistry, physics andmicrobiology, Volume 2: Major Cheese groups, second edition, page337-341, Chapman & Hall.

[0096] Sequence Listing and Deposited Microorganisms

[0097] The present application contains information in the form of asequence listing, which is appended to the application and alsosubmitted on a data carrier accompanying this application. In addition,the present application refers to deposited microorganisms. The contentsof the data carrier and the deposited microorganisms are fullyincorporated herein by reference.

[0098] Deposit of Biological Material

[0099] The following biological material has been deposited under theterms of the Budapest Treaty with the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 B, D-38124Braunschweig, Germany, and given the following accession number: DepositAccession Number Date of Deposit E. coli DSM 15441 12 Feb. 2003 E. coliDSM 15442 12 Feb. 2003

[0100] Materials And Methods

[0101] Media and substrates

[0102] Medium YP+2% G

[0103] 10 g yeast extract

[0104] 20 g peptone

[0105] water to 1 L

[0106] autoclave at 121° C., 20 minutes

[0107] add 100 ml 20% sterile glucose solution

[0108] RA Sporulation Medium:

[0109] 50 g succinic acid

[0110] 12.1 g sodium nitrate

[0111] 1 g glucose

[0112] 20 ml 50×Vogel's salts (Davis, R. H. and F. J. de Serres (1970),Meth. Enzymol. 17A: 79-143)

[0113] components are blended in one liter distilled water and filtersterilized

[0114] Britton Robinson buffer

[0115] 0.023 M phosphoric acid

[0116] 0.023 M acetic acid

[0117] 0.023 M boric acid

[0118] Titrated with NaOH or HCl to desired pH

[0119] Methods

[0120] Phospholipase Activity (LEU)

[0121] Lecithin is hydrolyzed under constant pH and temperature, and thephospholipase activity is determined as the rate of titrant (0.1N NaOH)consumption during neutralization of the liberated fatty acid.

[0122] The substrate is soy lecithin (L-α-Phosphotidyl-Choline), and theconditions are pH 8.00, 40.0° C., reaction time 2 min. The unit isdefined relative to a standard.

EXAMPLES Example 1: Expression of a Phospholipase A2 from Tuber albidumin Aspergillus oryzae

[0123] The DNA sequence disclosed in Soragni et al. (supra) was used todesign primers for PCR amplification of TbSP1 from genomic DNA, withappropriate restriction sites added to the primer ends to facilitatecloning of the PCR product (SEQ ID NO: 13 and 14). A Tuber albidumstrain, CBS 272.72, was obtained from the CBS (Centraalbureau voorSchimmelcultures, Utecht, The Netherlands), and cultured on X-agar at20° C., as recommended by the CBS in List of Cultures, 1996. Myceliumwas removed from the surface of the plate, and total DNA was isolatedusing a FastDNA Spin Kit (Bl0101, Inc., Vista, Calif.), following themanufacturer's instrucions. PCR amplification was performed usingExtensor Hi-Fidelity PCR Master Mix (ABgene, Surrey, U.K.) following themanufacturers instructions and using an annealing temperature of 52° C.for the first 5 cycles and 62° C. for the last 25 cycles. A single PCRproduct was obtained, and the sequence was determined and is presentedas SEQ ID 9 excluding the added synthetic restriction sites. Comparisonof this genomic sequence to the cDNA sequence presented by E. Soragni etal. revealed a single intron. When the intron is removed, the nucleotidesequence rom T. albidum CBS272.72 is 92.5% identical to that from T.borchii ATCC 96540, the strain used by E. Soragni et al. Thecorresponding peptide predicted from the T albidum CBS272.72 genesequence is 93.8% identical to the peptide sequence reported by Soragniet al.

[0124] The PCR fragment was restricted with BamHI and XhoI and clonedinto the Aspergillus expression vector pMStr57 using standardtechniques. The expression vector pMStr57 contains the same elements aspCaHj483 (WO 98/00529), with minor modifications made to the AspergillusNA2 promoter, and has sequences for selection and propogation in E.coli, and selection and expression in Aspergillus. Specifically,selection in Aspergillus is facilitated by the amdS gene of Aspergillusnidulans, which allows the use of acetamide as a sole nitrogen source.Expression in Aspergillus is mediated by a modified neutral amylase II(NA2) promoter from Asergillus niger which is fused to the 5′ leadersequence of the triose phosphate isomerase (tpi) encoding-gene fromAspergillus nidulans, and the terminator from theamyloglucosidase-encoding gene from Aspergillus niger. The phospholipaseA2-encoding gene of the resulting Aspergillus expression construct,pMStr70, was sequenced and the sequence was compared to that determinedpreviously for the uncloned PCR fragment, SEQ ID 9. A single T to Cmutation was found 52 bp downstream of the stop codon.

[0125]Aspergillus oryzae was transformed with pMStr70 using standardtechniques describeed in Christensen, T. et al., (1988), Biotechnology6, 1419-1422. Transformants were cultured in YP+2% G medium shaken at275 RPM at 30° C. and expression of the Tuber phospholipase A2, TbPLA2,was monitored by SDS-PAGE.

[0126] Protein Characterization

[0127] SDS-PAGE revealed two bands, with approximate Mw of 25 and 16kDa. The supernatant was purified by ion exchange chromatography on aSP-sepharose column equilibrated with 50 mM Acetate-buffer, and elutedwith 1M NaCl pH 5.0. The two proteins eluted in two separate fractions.Protein concentration was determined using Protein Assay ESL from Roche.Activity was determined in the LEU assay. Mw Concentration ActivitySpecific activity kDa mg/ml LEU/ml LEU/mg Pool 1 23-25 1.32  61  46 Pool2 16 0.42 272 648

[0128] The proteins were subjected to N-terminal sequencing. TheN-terminal sequence of pool 1 (23-25 kDa band) was found to correspondto amino acids 32-50 of SEQ ID NO: 10. Blotting of pool 2 (16 kDa band)revealed two bands with N-terminal sequences corresponding to aminoacids 86-98 and 91-103, respectively. Mass spectral analysis of the twobands showed masses of 13934 and 14348 Da respectively, matching within5 Da of values calculated from the sequences of amino acids 86-210 and91-210 of SEQ ID NO: 10, respectively.

Example 2 Purification Procedure for two forms of T. albidium PLA2Expressed in Aspergillus oryzae.

[0129] In most fermentations of the Aspergillus oryzae transformantdescribed in Example I that produces the T. albidum PLA2, two forms ofthe enzyme were detected during purification. One form ran at 22-23 kDain SDS-PAGE and corresponds to the peptide reported by Soragni et al.(supra). Additionally, a new form was detected which ran at 16-17 kDa inSDS-PAGE and which has a high specific activity and a high isoelectricpoint. Purification of the 22-23 kDa peptide Fermentation supernatantcontaining phospholipase from T. albidium expressed in A. oryzae(prepared in Example 1) was sterile filtered using EKS filter purchasedfrom Seitz Schenk Bad Kreuznach, Bettringerstrasse 42, Germany D-73550,Waldstetten.

[0130] The sterile filtered supernatant was then adjusted to pH of 8 andionic strength under 4 mSi.

[0131] Anion Exchange Chromatography

[0132] First step of purification was carried out on anion exchangechromatography using 50 ml Fast flow Q™ sepharose column purchased fromAmersham Pharmacia. The column was prequilibrated with 50 mM Trisacetate buffer pH 8. The sterile filtered fermentation broth was thenapplied on the column and the column was washed with the same bufferuntil all unbound material was washed out.

[0133] Bound proteins were eluted with the same buffer containing 1 MSodium chloride pH 8 with flow rate of 5 ml/minute and to a final volumeof 500 ml total buffer. Fractions of 5 ml each were collected usingfraction collector and Phospholipase activity of all fractionscontaining was assayed qualitatively using Lecithin as substrate usingL-α-Phosphatidyl choline purchased from Sigma product P-5638 andactivity was assayed using NEFA C kit purchased from Wako ChemicalsGmbH, Nissan Strasse 2, 41468 Neuss, Germany. Exact assay is describedbelow.

[0134] Substrate solutions containing 10 mg/ml of Lecithin substratewere prepared in different buffers such as 50 mM Acetate pH 5 or 50 mMHepes pH 7 or 50 mM Tris acetate pH 9 as buffers containing 2 mM CaCl2and 0.1% Triton X-100 purchased from Fluka chemicals. Substrate was thenemulsified by stirring and warming at 50° C. and then cooling to 40° C.and used as substrate.

[0135] Assay of activity was carried out using 300 μl of the substrateemulsion incubated with 25 μl of the enzyme fractions for 20 minutes at40° C. then 30 μl of the assay mixture was transferred to 300 μl of theNEFA C color reagent A prepared as described by the manufacturer andincubated for 10 minutes at 37° C. and 600 μl of the color reagent NEFAC B solution was added to the mixture and further incubated for 10minutes. The blue color formed was then measured in a spectrophotometerat 505 nm.

[0136] Protein Characterization

[0137] Fractions containing activity were then pooled and characterizedfor the molecular weight using SDS-PAGE electrophoresis using Novex Precasted gels 4 to 20% Tris-Glycine gels purchased from Invitrogen LifeTechnologies, Carlsbad Calif. 92008, USA. 22-23 kDa protein was detectedand blotted and N-terminal analysis was carried out using an AppliedBiosystem sequenator.

[0138] The first 19 amino acid residues from N-terminal were determinedand found to have the sequence of amino acids 32-50 of SEQ ID NO: 10.

[0139] Purification of the the 16-17 kDa Peptide

[0140] Sterile filtered fermentation supernatant of the T. albidumphospholiplase expressed in A. oryzae was adjusted to pH 4.7 and ionicstrength was adjusted below 4 mSi.

[0141] Cation Exchange Chromatography

[0142] SP-sepharose™ fast flow was purchased from Amersham Pharmacia. 50ml Column was packed and equilibrated with 50 mM acetate buffer pH 4.7the fermentation supernatant was then applied on column and unboundmaterial was washed using the same buffer.

[0143] Bound protein with high pl was then eluted with a linear saltgradient using 50 mM acetate buffer pH 4.7 containing 1 M Sodiumchloride. Fractions and flow rate were similar to those used for the lowpl form of the phospholipase. Phospholipase activity in the fractionswas assayed qualitatively using NEFA kit as above. Fractions containingPhospholipase activity were pooled and SDS-PAGE was carried out asdescribed above.

[0144] 16-17 kDa protein was observed which had a high isoelectricpoint, above 9.

[0145] The N-terminal analysis of the protein was carried out afterblotting the protein and using Applied biosystem sequentaor which showedan N-terminal which was completely different from the one published inSoragni et al. (supra). Thus, the T. albidum PLA2 was found to have twoforms deriving from differential N-terminal processing with N-terminalsequences corresponding to amino acids 86-105 and 91-110 of SEQ ID NO:10, respectively.

Example 3 Cheese Making with T. albidum Phospholipase

[0146] Pasteurized, non-homogenized cream (North Carolina StateUniversity Dairy Plant) was used to standardize five hundred gramspasteurized, non-homogenized skim milk (North Carolina State UniversityDairy Plant) to 3.5% fat thus producing full fat mozzarella cheese. Thecheese milk for each experiment was treated with either the 16-17 kD T.albidum phospholipase prepared according to example 2, or the commercialphospholipase Lecitase® 10 L (Novozymes A/S, Bagsvaerd, Denmark), andplaced in a 35° C. water bath until equilibrated to that temperature.The initial pH of the cheese milk was taken and 0.01% (w/w) of starterculture was added.

[0147] pH was monitored until a pH of 6.4 was reached. 250 pl rennet(Novozym 89 L) was diluted to in 9 ml total solution with deionisedwater, one ml of this solution was added to the cheese milk and thecheese milk was stirred vigorously for 3 minutes. The stir bar wasremoved and the rennetted milk was allowed to sit at 35° C.

[0148] After the above treatments, curd was ready to cut when a spatulawas inserted and sharp edges were seen. The cheese was cut by pushingthe cutter down and while holding the beaker quickly turning the cutterand finally pulling the cutter up. The curd was allowed to rest 5minutes then stirred gently with spoon. Temperature was raised to 41° C.with intermittent gentle agitation for ˜45 min or until the pH droppedto 6.0-5.9. The curd was drained using cheesecloth then replaced in thebeaker and kept at 41° C. in water bath while pouring off whey asneeded.

[0149] When the curd reached pH 5.3, the stainless steel bowl with thecurd in it was flooded in a water bath at 69° C. for 5 minutes then handstretched. Curd was tempered in cold icewater for 30 minutes. The cheesecurd was dried out with paper towel, weighed and refrigerated overnight.

[0150] Control cheese making experiments were made from the same batchof milk following the same procedures except that no phospholipase wasadded.

[0151] Actual cheese yield was calculated as the weight of cheese afterstretching relative to the total weight of cheese milk.

[0152] Moisture adjusted cheese yield was expressed as the actual yieldadjusted to standard constant level of moisture. Moisture adjusted yieldwas calculated by multiplying the actual yield and the ratio of actualmoisture content to standard moisture, according to the followingformula:

Y _(adj)=(Y _(act)×1−M _(act))/(1−M _(std))

[0153] where Y_(adj)=moisture adjusted cheese yield, Y_(act)=actualcheese yield, M_(act)=actual moisture fraction & M_(std)=standardmoisture fraction (0.48). The moisture adjusted cheese yield of allexperiments and controls are shown in table 1 TABLE 1 PhospholipaseYield increase mg enzyme Moisture adjusted compared to Treatmentprotein/g fat cheese yield control Control 0 10.72 T. albidum PLA2 0.05511.04 2.9% Control 0 11.25 T. albidum PLA2 0.055 11.57 2.8% Control 09.22 Lecitase ® 10L 0.18 9.48 2.7% Control 0 9.62 Lecitase ® 10L 0.189.90 2.8%

Example 4 Cloning and Expression of a Phospholipase (FvPLA2) fromFusarium venenatum in Aspergillus oryzae

[0154] Cells of the Fusarium venenatum A3/5 (originally deposited asFusarium graminearum ATCC 20334 and recently reclassified as Fusariumvenenatum by Yoder and Christianson, 1998, Fungal Genetics and Biology23: 62-80; and O'Donnell et al., 1998, Fungal Genetics and Biology 23:57-67) were grown for two days in Vogel's minimal medium (Davis, R. H.and F. J. de Serres (1970), Meth. Enzymol. 17A: 79-143) at 28° C. inshaking culture, filtered on sterile Miracloth (Calbiochem, San Diego,Calif., USA), and transferred to “RA sporulation medium” in which theywere incubated in shaking culture for an additional 24 hr at 28° C.Cells and spores were collected by centrifugation and lysed, and RNA wasextracted and transcribed into cDNA that was cloned into pZErO-2 by themethods described in WO 00/56762. The number of independent clones inthis library before amplification was 2.5×105, of which 92% containedinserts ranging in size from 550-2500 bp. Partial DNA sequences weredetermined for approximately 1000 randomly chosen clones and thesequences were stored in a computer database by methods described in WO00/56762.

[0155] The nucleotide sequence of a cDNA encoding TbSP1, a phospholipaseA2 from Tuber borchii, and the corresponding peptide translation werereported by E. Soragni et al., 2001. This translated peptide sequencewas compared to translations of the Fusarium venenatum partial cDNAsequences using the TFASTXY program, version 3.3t08 (Pearson et al.,1997). One translated F. venenatum sequence was identified as having 42%identity to TbSP1 through a 125 amino acid overlap. The completesequence of the cDNA insert of the corresponding clone, FM0700, wasdetermined and is presented as SEQ ID NO: 15, and the peptide translatedfrom this sequence, FvPLA2, is presented as SEQ ID NO: 16. This sequencewas used to design the primers FvPLA1 and FvPLA2.2 for PCR amplificationof the FvPLA2 encoding-gene from FM0700, with appropriate restrictionsites added to the primer ends to facilitate sub-cloning of the PCRproduct. FvPLA1: CTGGGATCCTCAAGATGAAGTTCAGCG FvPLA2.2:GACCTCGAGACCCGCCATTTAAGATT

[0156] PCR amplification was performed using Extensor Hi-Fidelity PCRMaster Mix (ABgene, Surrey, U.K.) following the manufacturersinstructions and using an annealing temperature of 52° C. and anextension temperature of 60° C. for 20 cycles.

[0157] The PCR fragment was restricted with BamHI and XhoI and clonedinto the Aspergillus expression vector pMStr57 using standardtechniques. The expression vector pMStr57 contains the same elements aspCaHj483 (WO 98/00529), with minor modifications made to the AspergillusNA2 promoter as described for the vector pMT2188 in WO 01/12794, and hassequences for selection and propagation in E. coli, and selection andexpression in Aspergillus. Specifically, selection in Aspergillus isfacilitated by the amdS gene of Aspergillus nidulans, which allows theuse of acetamide as a sole nitrogen source. Expression in Aspergillus ismediated by a modified neutral amylase II (NA2) promoter fromAspergillus niger which is fused to the 5′ leader sequence of the triosephosphate isomerase (tpi) encoding-gene from Aspergillus nidulans, andthe terminator from the amyloglucosidase-encoding gene from Aspergillusniger. The phospholipase-encoding gene of the resulting Aspergillusexpression construct, pMStr77, was sequenced and the sequence agreedcompletely with that determined previously for the insert of FM0700.

[0158] The Aspergillus oryzae strain BECh2 (WO 00/39322) was transformedwith pMStr77 using standard techniques (T. Christensen et al., 1988).Transformants were cultured in YP+2% G medium shaken at 275 RPM at 30°C. and expression of FvPLA2 was monitored by SDS-PAGE.

[0159] A strain of Eschericia coli containing a gene encoding thephospholipase from F. venenatum was deposited by the inventors under theterms of the Budapest Treaty with Deutsche Sammlung von Microorganismenund Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig,Germany. The deposit date was 12 Feb. 2003, and the accession number wasDSM 15442.

Example 5 Purification and Sequence Comparison of FvPLA2

[0160] FvPLA2 from the fermentation of example 4 was purified by ionexchange chromatography on a SP-sepharose column equilibrated with 50 mMAcetate-buffer pH 4.7, and eluted with 1M NaCl pH 4.7. Fractions wereanalyzed on SDS-PAGE, and fractions containing a 14 kDa protein werepooled. The identity of the pure protein was confirmed by determiningthe the N-terminal sequence, which was identical to the sequence fromamino acid (aa) 29-40 of SEQ ID NO: 16. Additionally, the mass of thepeptide was determined by mass spectral analysis, because the apparentsize estimated from SDS-PAGE, 14 kDa, is smaller than that of thepeptide predicted by processing the theoretical peptide in SEQ ID NO:16. The mass of the purified, active FvPLA2 was found to be 13336 Da.This molecular mass indicates additional processing at the C-terminus,and is consistent with a cleavage between amino acids 149 and 150 in SEQID NO: 16, as the peptide sequence from amino acid 29 to 149 has atheoretical mass of 13335,66 Da.

[0161] A comparison of the mature processed peptide (amino acids 29-149of SEQ ID NO: 16) with known sequences showed that the closest prior-artsequence was a phospholipase from Verticillium dahliae translated fromUnisequence ID: VD0100C34 from the COGEME Phytopathogenic Fungi andOomycete EST Database Version 1.2 (http://cogeme.ex.ac.uk/) (Soanes etal. (2002) Genomics of phytopathogenic fungi and the development ofbioinformatic resources. Mol Plant Microbe Interact. 15(5): 421-7). Theprocessing of the partial peptide predicted from the V. dahliae sequencewas estimated by comparison to the found processing for FvPLA2. Theidentity between amino acids 29 to 149 of SEQ ID NO: 16 and theestimated sequence of the mature peptide of the V. dahliae phospholipasewas calculated to be 77%.

Example 6 Physical Properties of FvPLA2

[0162] Catalytic Activity

[0163] Phospholipase activity as a function of enzyme concentration wasdetermined in the LEU assay for FvPLA2 of example 4. Results are shownin table 1. TABLE 1 Enzyme conc. LEU (μg/ml) (μeq NaOH/min) 71.1 14.053.3 12.7 21.3 10.6 10.7 7.4 5.3 5.6 2.7 4.1

[0164] Temperature Profile

[0165] The enzyme activity as a function of temperature was determinedfor an enzyme solution with a concentration of 5.3 μg/ml. Otherconditions as in the LEU assay. Results are shown in table 2. TABLE 2Temperature LEU (° C.) (μeq NaOH/min) 25 3.10 35 4.87 40 5.41 45 6.97 507.86 55 9.03 60 8.27 65 6.90

[0166] pH Stability

[0167] The enzyme was diluted in a Britton Robinson buffer at thespecified pH for 30 min at 30° C. After further dilution in watercatalytic activity was measured in the LEU assay. Results are shown intable 3. TABLE 3 pH LEU (μeq NaOH/min) 2 3.78 3 5.11 4 5.60 5 5.49 65.37 7 5.61 8 5.52 9 5.64 10  5.50 11  5.21

[0168] Thermo Stability

[0169] The enzyme was diluted in Britton Robinson buffer at pH 3 and 10respectively, and at pH7 with 30% sorbitol. After incubation at thespecified temperature for 30 minutes, the solution was cooled to thereaction temperature and assayed in the LEU assay. The results are shownin table 4; activities are given relative to the highest measuredactivity. TABLE 4 Relative activity (%) as a function of pH andtemperature Temperature (° C.) pH 3 pH 10 pH7/30% sorbitol 30 100% 100% 87% 40  95%  92% 100% 50  16%  14%  68% 60  1%  0%  2%

Example 7 Cheese Making with FvPLA2

[0170] Pasteurized, non-homogenized cream (North Carolina StateUniversity Dairy Plant) was used to standardize five hundred gramspasteurized, non-homogenized skim milk (North Carolina State UniversityDairy Plant) to 3.5% fat thus producing full fat mozzarella cheese.

[0171] The cheese milk for each experiment was treated with either theF. venenatum phospholipase (FvPLA2) prepared according to example 5, orof the commercial phospholipase Lecitase® 10 L (Novozymes A/S,Bagsvaerd, Denmark), and placed in a 35° C. water bath untilequilibrated to that temperature. The initial pH of the cheese milk wastaken and 0.01% (w/w) of starter culture at was added.

[0172] pH was monitored until a pH of 6.4 was reached. 250 μl rennet(Novozym 89 L) was diluted to in 9 ml total solution with deionizedwater, one ml of this solution was added to the cheese milk and thecheese milk was stirred vigorously for 3 minutes. The stir bar wasremoved and the rennetted milk was allowed to sit at 35° C.

[0173] After the above treatments, curd was ready to cut when a spatulawas inserted and sharp edges were seen. The cheese was cut by pushingthe cutter down and while holding the beaker quickly turning the cutterand finally pulling the cutter up. The curd was allowed to rest 5minutes then stirred gently with spoon. Temperature was raised to 41° C.with intermittent gentle agitation for ˜45 min or until the pH droppedto 6.0-5.9. The curd was drained using cheese-cloth then replaced in thebeaker and kept at 41° C. in water bath while pouring off whey asneeded.

[0174] When the curd reached pH 5.3, the stainless steel bowl with thecurd in it was flooded in a water bath at 69° C. for 5 minutes then handstretched. Curd was tempered in cold icewater for 30 minutes. The cheesecurd was dried out with paper towel, weighed and refrigeratedover-night.

[0175] Control cheese making experiments were made from the same batchof milk following the same procedures except that no phospholipase wasadded.

[0176] Actual cheese yield was calculated as the weight of cheese afterstretching relative to the total weight of cheese milk.

[0177] Moisture adjusted cheese yield was expressed as the actual yieldadjusted to standard constant level of moisture. Moisture adjusted yieldwas calculated by multiplying the actual yield and the ratio of actualmoisture content to standard moisture, according to the followingformula:

Yadj=Yact×(1−Mact)/(1−Mstd)

[0178] where Yadj=moisture adjusted cheese yield, Yact=actual cheeseyield, Mact=actual moisture fraction & Mstd=standard moisture fraction(0.48).

[0179] The moisture adjusted cheese yield of all experiments andcontrols are shown in table 5. TABLE 5 Phospholipase Yield increase mgenzyme Moisture adjusted compared to Treatment protein/g fat cheeseyield control Control 0 11.70 FvPLA2 0.071 11.95 2.1% Control 0 11.50FvPLA2 0.071 11.83 2.8% Control 0 9.22 Lecitase ® 10L 0.18 9.48 2.7%Control 0 9.62 Lecitase ® 10L 0.18 9.90 2.8%

Example 8 Cheese Making with FvPLA2

[0180] Milk was pasteurized at 72° C. for 15 seconds and then cooled tobelow 10° C. Milk was standardized to 2.4% fat with cream. Afterstandardization the milk was preheated in a heat ex-changer at apre-ripening temperature of 34.5° C. 150 kg milk was poured into eachcheese vat and 15 g culture (F-DVS ST-M6) was added. The phospholipasefrom example 5 was added in a dosage of 5 LEU/g fat and the milk wasincubated for 1 h at 34.5° C. Rennet (Chy-Max Plus, 200 IMCU) was addedand agitation was continued for not more than 4 min.

[0181] After approx. 60 min when the coagulum was judged ready it wascut using 10 mm knives. The agitator was returned to the vat and after10 min. the scalding was started by increasing the temperature to 41° C.within 30 min. After reaching 41° C. a further stirring forapproximately 20 min. took place until a titratable acidity of0.15-0.16% was reached. The curd was allowed to settle in the vat, andwhey was drained. The curd was cut in uniform blocks and the blocks wereturned and stacked into two. Subsequently, at intervals of 10 min. theblocks were turned and kept in stacks of two. At a pH of around5.15-5.20, the curd was milled in a milling machine. The curd pieceswere added two percent of salt (weight/weight).

[0182] After milling all the curd was added into the stretcher, whichcontains 70 l preheated water at 74° C. Around 20 l of hot water wastransferred to the upper chamber and the cheese is added. When the curdtemperature reached 62° C., the stretching was stopped and the curdmoved to the extruder. Cheeses were extruded into 8-9 cheese loaves,each of 2.3 kg, and cooled in 5-7° C. water for 20 min. After 20 min.cooling the cheeses were moved to the saturated brine and brined for 1.5hours at 5-6° C. The brine was made by blending 120 kg water, addingsalt to 22Be, 750 g CaCl2 (34% solution) and adjusted to pH 5.1. Afterbrining each cheese was dried for around 30 min. and weighed beforevacuum packaging. Samples were taken for pH and compositional analyses(moisture, salt, fat and protein) after about 1 week's storage in coldroom.

[0183] Actual yield (AY) was adjusted to 48% moisture in cheese:${{Adj}\quad {Yield}} = \frac{{AY} \times \left( {100 - {\% \quad {moisture}}} \right)}{100 - 48}$

TABLE 6 Adjusted yield Adjusted yield (kg) (kg) Average yield YieldControl Experimental increase (kg) increase (%) Day 1 10.62 10.81 10.7010.90 0.195 1.8 Day 2 9.90 10.16 9.95 10.14 0.225 2.3 Day 3 10.00 10.1510.01 10.16 0.15 1.5

Example 9 Over-expression of Aspergillus oryzae PLA2 (AoPLA2) inAspergillus oryzae

[0184] Medium

[0185] DAP2C-1

[0186] 11g MgSO₄.7H₂O

[0187] 1 g KH₂PO₄

[0188] 2 g Citric acid, monohydrate

[0189] 30 g maltodextrin

[0190] 6 g K₃PO₄.3H₂₀

[0191] 0.5 g yeast extract

[0192] 0.5 ml trace metals solution

[0193] 1 ml Pluronic PE 6100 (BASF, Ludwigshafen, Germany)

[0194] Components are blended in one liter distilled water and portionedout to flasks, adding 250 mg CaCO3 to each 150 ml portion.

[0195] The medium is sterilized in an autoclave. After cooling thefollowing is added to 1 liter of medium:

[0196] 23 ml 50% w/v (NH₄)₂HPO₄, filter sterilized

[0197] 33 ml 20% lactic acid, filter sterilized

[0198] Trace Metals Solution

[0199] 6.8 g ZnCl₂

[0200] 2.5 g CuSO₄.5H₂₀

[0201] 0.24 g NiCl₂.6H₂₀

[0202] 13.9 g FeSO₄.7H₂₀

[0203] 8.45 g MnSO₄.H₂₀

[0204] 3 g Citric acid, monohydrate

[0205] Components are blended in one liter distilled water.

[0206] The cloning and partial sequencing of a cDNA encoding aphospholipase A2 from Aspergillus oryzae is described in WO 00/56762.The full sequence of the clone, AS3812, is given in SEQ ID NO: 6.

[0207] This sequence was used to design the primer AoPLA1 for use withthe vector primer pYESrev in PCR amplification of the PLA2 encoding-genefrom AS3812 with the addition of a restriction site to facilitatesub-cloning of the PCR product: AoPLA1: TGAGGATCCATCATGAAGAACATCTTCGpYESrev: gggcgtgaatgtaagcgtgac

[0208] PCR amplification was accomplished using Extensor Hi-Fidelity PCRMaster Mix (AB-gene, Surrey, U.K.) following the manufacturersinstructions and using an annealing temperature of 52° C. for the first5 cycles and 62° C. for the last 25 cycles, and an extension time of 1.5minutes.

[0209] The PCR fragment was restricted with BamHI and XhoI and clonedinto the Aspergillus expression vector pMStr57 (described in Example 1)using standard techniques. The phospholipase-encoding gene of theresulting Aspergillus expression construct, pMStr71, was sequenced andthe sequence agreed completely with that determined previously for theinsert of AS3812.

[0210] The Aspergillus oryzae strain BECh2 (WO 00/39322) was transformedwith pMStr71 using standard techniques (T. Christensen et al., 1988).Transformants were cultured in DAP2C-1 medium shaken at 270 RPM at 37°C. for 4 days and expression of phospholipase was monitored by SDS-PAGE.

Example 10 Purification and Determination of Peptide Processing

[0211] The Aspergillus oryzae phospholipase from the fermentation ofexample 9 was filtered through 0.22μ sterile filter Seitz-EKS obtainedfrom Pall Corporation (Pall SeitzSchenk Filter Systems GmbH PianigerStr.137 D-55543 Bad Kreuznach, Germany). The sterile filtered solutionwas then adjusted to pH 4.7 using dilute acetic acid. Ionic strength ofthe fermentation supernatant was then adjusted so that saltconcentration was low and ionic strength was under 4 mSi. Purificationof the desired PLA2 protein was obtained by cation exchangechromatography using SP sepharose fast Flow matrix obtained fromAmersham-Pharmacia (Sweden). The cation exchanger matrix was packedwashed and pre-equilibrated with 50 mM Sodium acetate buffer pH 4.7(Buffer A) on XK26 column obtained from Amersham Pharmacia. Fermentationsupernatant containing the desired PLA2 adjusted for pH and ionicstrength was then applied on the column. Unbound material was thenwashed with the buffer A until all the UV absorbing material was washedout, which was monitored by UV detector attached to fraction collectorequipment. Bound proteins were then eluted with a linear salt gradientusing Buffer B, which contained 1 M Sodium chloride as salt in 50 mMSodium acetate buffer pH 4.7. Total volume of the linear gradientreaching 1 M salt concentration was around 500 ml (10 column volume).Fractions of 10 ml each were collected during the elution. All thefractions were assayed for phospholipase activity using Lecithin assubstrate obtained from Sigma chemicals. Fatty acids released fromLecithin on incubation with the phospholipase were detected using NEFA Ckit obtained from Waco chemicals. Fractions containing phospholipaseactivity were then checked for purity of the protein using standardSDS-PAGE technique. Fractions were pooled that contained a single bandof the desired PLA2 showing molecular weight of around 16 kDa, asdetermined by comparison to molecular weight standards fromAmersham-Pharmacia.

[0212] The identity of the pure protein was confirmed by determining theN-terminal sequence, which was identical to the sequence from amino acid(aa) 37-45 of SEQ ID NO: 7. Additionally, the mass of the peptide wasdetermined by mass spectral analysis. The purified, active AspergillusPLA2 gave two masses, 14114 and 14242 Da. These molecular massesindicate additional processing at the C-terminus, consistent withcleavage between amino acids 121 and 122 in SEQ ID NO: 7, as the peptidesequence from amino acid 37 to 121 has a theoretical mass of 14114.11 Daand cleavage between amino acids 122 and 123, predicting the peptidesequence from amino acid 37 to 123 with a theoretical mass of 14242.29Da.

Example 11 Expression of Incompletely Processed Phospholipase fromAspergillus oryzae and Fusarium venenatum

[0213] Processing of the Aspergillus oryzae PLA2 (AoPLA2) and theFusarium venenatum PLA (FvPLA2) at both the N- and C-termini occurs atsingle or multiple basic residues (lys or arg), typical of the cleavagesites of the Kexin-like maturases, which are often responsible forprocessing propeptides (Jalving, R., et al. (2000) Appl. Environ.Microbiol. 66: 363-368). In order to determine the effect of processingon the activity of AoPLA2 and FvPLA2, the enzymes were expressed in aKexin deficient strain of Aspergillus oryzae. Processing was thenassessed by SDS-PAGE, and phospholipase activity was measured forcultures of strains expressing AoPLA2 and FvPLA2 in both wild-type andKexin deficient backgrounds.

[0214] A Kexin deficient strain of Aspergillus oryzae (kexB⁻) wasconstructed by a disrupting the kexB gene of A. oryzae (EMBL:AB056727)by methods established in the art, such as those described in WO98/12300 and U.S. Pat. No. 6,013,452. Disruption of kexB was confirmedby Southern blot analysis and by monitoring the expression of peptideswhere KexB is known to be responsible for maturation. The kexB⁻ strainwas transformed with the AoPLA2 expression construct described inExample 9, and with the FvPLA2 expression construct described in Example4. These strains were fermented in YP+2% G at 30° C., along with thekexB⁺ expression strains for both AoPLA2 and FvPLA2 described inExamples 9 and 4, and untransformed strains as controls. AoPLA2expressing strains were shaken at 200 RPM for 4 days while FvPLA2expressing strains were shaken at 275 RPm for 3 days. Phospholipaseexpression and processing were assessed by SDS-PAGE.

[0215] In SDS-PAGE analysis, AoPLA2 was resolved as a distinct singleband in both kexB⁺ and kexB⁻ strains. When expressed in the kexB⁺strain, AoPLA2 ran at ca. 16 kDa, consistent with the migration observedearlier for fully processed AoPLA2 (Example 10), while in the kexB⁻strain, AoPLA2 ran at ca. 27-28 kDa, consistent with a lack ofprocessing or incomplete processing. When expressed in the kexB⁺ strainFvPLA2 was resolved as two bands with apparent molecular weights of 17kDa and 14 kDa. The 14 kDa band corresponds to the fully processedpeptide (Example 5), while the 17 kDa peptide is a partially processedform. When expressed in the kexB⁻ strain, FvPLA2 ran as a single band atca. 18-19 kDa, a size consistent with incomplete processing. No similarbands were seen in any of the control samples from untransformedstrains. Relative band intensities suggest that expression of AoPLA2 inthe kexB⁻ strain was 1/5 to 1/10 the level of that in the kexB⁺ strain,while expression of FvPLA2 in the kexB⁻ strain was the same to 1/2 thelevel of that in the kexB⁺ strain.

[0216] The activity of the phospholipases produced by each strain wasdetermined in the LEU assay and is shown in table 7. TABLE 7 Straingenotype Activity KexB FvPLA AoPLA2 LEU/ml + − − 0 − − − 0 + + − 38  − +− 0 + − + 56  − − + 0

[0217]

1 16 1 211 PRT Tuber borchii 1 Met Val Lys Ile Ala Ala Ile Ile Leu LeuMet Gly Ile Leu Ala Asn 1 5 10 15 Ala Ala Ala Ile Pro Val Ser Glu ProAla Ala Leu Asn Lys Arg Gly 20 25 30 Asn Ala Glu Val Ile Ala Glu Gln ThrGly Asp Val Pro Asp Phe Asn 35 40 45 Thr Gln Ile Thr Glu Pro Thr Gly GluGly Asp Arg Gly Asp Val Ala 50 55 60 Asp Glu Thr Asn Leu Ser Thr Asp IleVal Pro Glu Thr Glu Ala Ala 65 70 75 80 Ser Phe Ala Ala Ser Ser Val SerAla Ala Leu Ser Pro Val Ser Asp 85 90 95 Thr Asp Arg Leu Leu Tyr Ser ThrAla Met Pro Ala Phe Leu Thr Ala 100 105 110 Lys Arg Asn Lys Asn Pro GlyAsn Leu Asp Trp Ser Asp Asp Gly Cys 115 120 125 Ser Lys Ser Pro Asp ArgPro Ala Gly Phe Asn Phe Leu Asp Ser Cys 130 135 140 Lys Arg His Asp PheGly Tyr Arg Asn Tyr Lys Lys Gln His Arg Phe 145 150 155 160 Thr Glu AlaAsn Arg Lys Arg Ile Asp Asp Asn Phe Lys Lys Asp Leu 165 170 175 Tyr AsnGlu Cys Ala Lys Tyr Ser Gly Leu Glu Ser Trp Lys Gly Val 180 185 190 AlaCys Arg Lys Ile Ala Asn Thr Tyr Tyr Asp Ala Val Arg Thr Phe 195 200 205Gly Trp Leu 210 2 588 DNA Verticillium dahliae misc_feature (25)..(26) nis a, c, g, or t 2 cagtttgaag tcccagcccc tgctnntcct cctgcttctccccgtccagt ctttgggatt 60 ttcctctcat c atg aag ttc aac gca att ctc ctggcc ctc gtg cct gcc 110 Met Lys Phe Asn Ala Ile Leu Leu Ala Leu Val ProAla 1 5 10 gcc ctg gct ctg ccc acc acc gac gag gcg cag acc ccc aag ctcgcc 158 Ala Leu Ala Leu Pro Thr Thr Asp Glu Ala Gln Thr Pro Lys Leu Ala15 20 25 gcg cgc cag agc atc acg gcc gtc acc gac agc ctg tcc ttc tcc ctg206 Ala Arg Gln Ser Ile Thr Ala Val Thr Asp Ser Leu Ser Phe Ser Leu 3035 40 45 acg ctg cct cag ttc acc acg cgc cgc aac aac cgc aac ccc gcc aac254 Thr Leu Pro Gln Phe Thr Thr Arg Arg Asn Asn Arg Asn Pro Ala Asn 5055 60 ctc gac tgg agc tcc gac ggc tgc aca acg tct cct gac aac cca ttc302 Leu Asp Trp Ser Ser Asp Gly Cys Thr Thr Ser Pro Asp Asn Pro Phe 6570 75 gga ttc ccc ttt gtg ccg gcc tgc cac cgc cac gac ttt ggc tac cac350 Gly Phe Pro Phe Val Pro Ala Cys His Arg His Asp Phe Gly Tyr His 8085 90 aac ttc cgc gcc cag acc cgc ttc acc gag agc aac aag ctc cgc atc398 Asn Phe Arg Ala Gln Thr Arg Phe Thr Glu Ser Asn Lys Leu Arg Ile 95100 105 gac aac cag ttc agg acc gat ctg agg ttc cag tgc cag tct tcg agc446 Asp Asn Gln Phe Arg Thr Asp Leu Arg Phe Gln Cys Gln Ser Ser Ser 110115 120 125 gtg cgc ggc gtg tgc aac gcc ctg gcg gac gtc tac tac tct gccgtc 494 Val Arg Gly Val Cys Asn Ala Leu Ala Asp Val Tyr Tyr Ser Ala Val130 135 140 cgg gcg ttc ggc ggt gac gac gcc acc ccc ggc aag agg gac gagcac 542 Arg Ala Phe Gly Gly Asp Asp Ala Thr Pro Gly Lys Arg Asp Glu His145 150 155 tcg gaa ctc gtc ggc atc tac gac gag aag gtc ggc atc tac gata 588 Ser Glu Leu Val Gly Ile Tyr Asp Glu Lys Val Gly Ile Tyr Asp 160165 170 3 172 PRT Verticillium dahliae 3 Met Lys Phe Asn Ala Ile Leu LeuAla Leu Val Pro Ala Ala Leu Ala 1 5 10 15 Leu Pro Thr Thr Asp Glu AlaGln Thr Pro Lys Leu Ala Ala Arg Gln 20 25 30 Ser Ile Thr Ala Val Thr AspSer Leu Ser Phe Ser Leu Thr Leu Pro 35 40 45 Gln Phe Thr Thr Arg Arg AsnAsn Arg Asn Pro Ala Asn Leu Asp Trp 50 55 60 Ser Ser Asp Gly Cys Thr ThrSer Pro Asp Asn Pro Phe Gly Phe Pro 65 70 75 80 Phe Val Pro Ala Cys HisArg His Asp Phe Gly Tyr His Asn Phe Arg 85 90 95 Ala Gln Thr Arg Phe ThrGlu Ser Asn Lys Leu Arg Ile Asp Asn Gln 100 105 110 Phe Arg Thr Asp LeuArg Phe Gln Cys Gln Ser Ser Ser Val Arg Gly 115 120 125 Val Cys Asn AlaLeu Ala Asp Val Tyr Tyr Ser Ala Val Arg Ala Phe 130 135 140 Gly Gly AspAsp Ala Thr Pro Gly Lys Arg Asp Glu His Ser Glu Leu 145 150 155 160 ValGly Ile Tyr Asp Glu Lys Val Gly Ile Tyr Asp 165 170 4 185 PRT Neurosporacrassa 4 Met Lys Phe Phe Ser Ala Leu Ala Leu Ser Ser Leu Leu Pro Thr Ala1 5 10 15 Ala Trp Ala Trp Thr Gly Ser Glu Ser Asp Ser Thr Gly Ala AspSer 20 25 30 Leu Phe Arg Arg Ala Glu Thr Ile Gln Gln Thr Thr Asp Arg TyrLeu 35 40 45 Phe Arg Ile Thr Leu Pro Gln Phe Thr Ala Tyr Arg Asn Ala ArgSer 50 55 60 Pro Ala Thr Leu Asp Trp Ser Ser Asp Ser Cys Ser Tyr Ser ProAsp 65 70 75 80 Asn Pro Leu Gly Phe Pro Phe Ser Pro Ala Cys Asn Arg HisAsp Phe 85 90 95 Gly Tyr Arg Asn Tyr Lys Ala Gln Ser Arg Phe Thr Asp AsnAsn Lys 100 105 110 Leu Lys Ile Asp Gly Asn Phe Lys Thr Asp Leu Tyr TyrGln Cys Asp 115 120 125 Thr His Gly Tyr Gly Ser Thr Cys His Ala Leu AlaAsn Val Tyr Tyr 130 135 140 Ala Ala Val Arg Glu Phe Gly Arg Thr Lys GlyGlu Leu Gln Glu Glu 145 150 155 160 Tyr Asp Leu Leu Leu Ala His Tyr AsnGlu Leu Val Ala Glu Ala Ile 165 170 175 Ala Lys Gly Glu Asp Pro Leu TyrTyr 180 185 5 169 PRT Helicosporium sp. 5 Met Lys Ser Phe Thr Phe ValVal Leu Ala Leu Leu Pro Phe Ser Ser 1 5 10 15 Ala Leu Pro Phe Gly LeuPhe His Arg Gly Gly Ile Ala Ser Arg Ala 20 25 30 Thr Ile Glu Glu Thr ThrAsp Thr Leu Leu Phe Ser Thr Pro Ile Ala 35 40 45 Gln Phe Glu Ala Ala ArgAsn Ala Gln Asn Pro Ser Thr Leu Asp Trp 50 55 60 Ser Ser Asp Gly Cys SerSer Ser Pro Asp Asp Pro Phe Gly Phe Asp 65 70 75 80 Phe Leu Ser Ser CysHis Arg His Asp Phe Gly Tyr Arg Asn Tyr Lys 85 90 95 Lys Gln Asn Arg PheThr Ala Pro Asn Lys Ala Arg Ile Asp Thr Asn 100 105 110 Phe Lys Thr AspMet Tyr Asn Gln Cys Asn Thr Glu Ser Asn Ile Phe 115 120 125 Thr Arg AlaAla Cys Lys Ala Val Ala Asp Ile Tyr Tyr Glu Ala Val 130 135 140 Lys ThrPhe Gly Ser Lys Lys Arg Ala Ala Glu Ala Leu Ala Ala Arg 145 150 155 160Gln Met Glu Glu Asn Val Ala Lys Ala 165 6 942 DNA Aspergillus oryzae CDS(61)..(726) 6 cgcaagcatc acatctactt cttattgcct attctgtccg agtgctagccacttatcatc 60 atg aag aac atc ttc gtt gcc act ttg ggc ctg ttc gcc gcagtt tcg 108 Met Lys Asn Ile Phe Val Ala Thr Leu Gly Leu Phe Ala Ala ValSer 1 5 10 15 tct gcc ttg ccc tac aca acc cct gtc aat gac aat ccc atctct gct 156 Ser Ala Leu Pro Tyr Thr Thr Pro Val Asn Asp Asn Pro Ile SerAla 20 25 30 tta caa gca cgc gcg aca aca tgc tcg gcc aag gcc acg gat aacctc 204 Leu Gln Ala Arg Ala Thr Thr Cys Ser Ala Lys Ala Thr Asp Asn Leu35 40 45 atc ttc aag gtc tcc atg aag acc ttc cag aag gcg cgc aag gcc aag252 Ile Phe Lys Val Ser Met Lys Thr Phe Gln Lys Ala Arg Lys Ala Lys 5055 60 aac ccc tcc aag tgc aac tgg tca tcg gac aac tgc tcc aag tca ccc300 Asn Pro Ser Lys Cys Asn Trp Ser Ser Asp Asn Cys Ser Lys Ser Pro 6570 75 80 gat aag ccc gat gga tac aac ttc atc ccc agc tgc caa aga cac gat348 Asp Lys Pro Asp Gly Tyr Asn Phe Ile Pro Ser Cys Gln Arg His Asp 8590 95 ttc ggc tac cgg aac acg aag aag cag aag cgc ttc aca aag gcc atg396 Phe Gly Tyr Arg Asn Thr Lys Lys Gln Lys Arg Phe Thr Lys Ala Met 100105 110 aag aag cgc att gac gac aac ttc aag aag gat ctc tac aag tac tgc444 Lys Lys Arg Ile Asp Asp Asn Phe Lys Lys Asp Leu Tyr Lys Tyr Cys 115120 125 agc caa ttc tcg ggc tgg agc tca tgg aag gga gtg gag tgc cgt cgc492 Ser Gln Phe Ser Gly Trp Ser Ser Trp Lys Gly Val Glu Cys Arg Arg 130135 140 ctt gcg gat gtc tac tat act gct gtc cgc cac ttt ggc aag cgt gat540 Leu Ala Asp Val Tyr Tyr Thr Ala Val Arg His Phe Gly Lys Arg Asp 145150 155 160 gaa gcg ctt gag ttt gac cct gag gtt gag ttc gag aag cgt gatgag 588 Glu Ala Leu Glu Phe Asp Pro Glu Val Glu Phe Glu Lys Arg Asp Glu165 170 175 gtg gcc gat gtc cag cct gac gaa ttt gat aac ttt gac ggt tctgaa 636 Val Ala Asp Val Gln Pro Asp Glu Phe Asp Asn Phe Asp Gly Ser Glu180 185 190 gtt gac cct gat atc gag ggc cag gtc att ccc gaa gtt ctt gaagat 684 Val Asp Pro Asp Ile Glu Gly Gln Val Ile Pro Glu Val Leu Glu Asp195 200 205 gat gga gtg gat gtg gag aac ctc gac gat att gaa aac ctg 726Asp Gly Val Asp Val Glu Asn Leu Asp Asp Ile Glu Asn Leu 210 215 220taggttttcg gcattggctc tacactttgc aaatgggtcg tcataatcca ttggaagccg 786gaggaggagg gaaatcaagg catcttttgg ttgtcagtaa ctttgagtgc ctagtttgtg 846aattgttttt tgaggttcta tttgaattct gcttttgttc aatcttatag cttcctacgt 906tgttgtcatt taaaaatgga caggagtatc tgtgag 942 7 222 PRT Aspergillus oryzae7 Met Lys Asn Ile Phe Val Ala Thr Leu Gly Leu Phe Ala Ala Val Ser 1 5 1015 Ser Ala Leu Pro Tyr Thr Thr Pro Val Asn Asp Asn Pro Ile Ser Ala 20 2530 Leu Gln Ala Arg Ala Thr Thr Cys Ser Ala Lys Ala Thr Asp Asn Leu 35 4045 Ile Phe Lys Val Ser Met Lys Thr Phe Gln Lys Ala Arg Lys Ala Lys 50 5560 Asn Pro Ser Lys Cys Asn Trp Ser Ser Asp Asn Cys Ser Lys Ser Pro 65 7075 80 Asp Lys Pro Asp Gly Tyr Asn Phe Ile Pro Ser Cys Gln Arg His Asp 8590 95 Phe Gly Tyr Arg Asn Thr Lys Lys Gln Lys Arg Phe Thr Lys Ala Met100 105 110 Lys Lys Arg Ile Asp Asp Asn Phe Lys Lys Asp Leu Tyr Lys TyrCys 115 120 125 Ser Gln Phe Ser Gly Trp Ser Ser Trp Lys Gly Val Glu CysArg Arg 130 135 140 Leu Ala Asp Val Tyr Tyr Thr Ala Val Arg His Phe GlyLys Arg Asp 145 150 155 160 Glu Ala Leu Glu Phe Asp Pro Glu Val Glu PheGlu Lys Arg Asp Glu 165 170 175 Val Ala Asp Val Gln Pro Asp Glu Phe AspAsn Phe Asp Gly Ser Glu 180 185 190 Val Asp Pro Asp Ile Glu Gly Gln ValIle Pro Glu Val Leu Glu Asp 195 200 205 Asp Gly Val Asp Val Glu Asn LeuAsp Asp Ile Glu Asn Leu 210 215 220 8 249 PRT Neurospora crassa 8 MetLys Pro Phe Phe Leu Ile Ser Leu Leu Val Thr Val Phe Met Ser 1 5 10 15Leu Met Leu Ala Thr Thr Ala Gln Pro Ser Leu Pro Leu Asn Asn Arg 20 25 30Arg Glu Leu Ala Glu His Pro Pro Val Lys Gly Asn Pro Pro Asn Thr 35 40 45Gly Tyr Ala Leu Asp Trp Cys Lys Tyr Thr Ala Gly Met Leu Phe Gln 50 55 60Trp Asp Leu Pro Thr Phe Ile Lys His Arg Glu Ala Asn Phe Ser Leu 65 70 7580 Gly Arg Leu Thr Trp Asp Trp Ser Ser Asp Gly Cys Thr His Val Pro 85 9095 Asp Asn Pro Val Gly Phe Pro Phe Lys Pro Ala Cys Gln Arg His Asp 100105 110 Phe Gly Tyr Arg Asn Tyr Gln Val Gln Phe His Phe Thr Pro Arg Ala115 120 125 Arg Trp Lys Ile Asp Glu Asn Phe Leu Lys Glu Met Lys Phe GlnCys 130 135 140 Ile Gly His Asn Ile Phe Asn Ala Cys His Phe Met Ala HisVal Tyr 145 150 155 160 His Trp Gly Val Arg Thr Phe Tyr Lys Gly His GluGln Tyr Arg Glu 165 170 175 Ser Glu Pro Ser His Lys Met Met Asp Thr MetVal Ala Ser Glu Ser 180 185 190 Ser Asp Val Phe Asp Gly Met Asp Ala AspGlu Ala Arg Asp Ala Leu 195 200 205 Asn Pro Tyr Leu Ser Glu Glu Lys ThrLys Glu Tyr Tyr Asp Arg Ala 210 215 220 Leu Ala Arg Tyr Asn Lys Cys ValGlu Glu Ala Met Ala Gln Gly Ile 225 230 235 240 Asp Leu Gln Lys Tyr TrpAla Ala Phe 245 9 832 DNA Tuber albidum CDS (2)..(426) CDS (476)..(680)9 a atg gtc aag att gct gcc att gtc ctc cta atg gga att cta gcc aat 49Met Val Lys Ile Ala Ala Ile Val Leu Leu Met Gly Ile Leu Ala Asn 1 5 1015 gct gcc gcc atc cct gtc agc gag cca gca gcc ctg gcg aag cgt gga 97Ala Ala Ala Ile Pro Val Ser Glu Pro Ala Ala Leu Ala Lys Arg Gly 20 25 30aac gct gag gtc att gct gaa caa act ggt gat gtc ccg gat ttc aac 145 AsnAla Glu Val Ile Ala Glu Gln Thr Gly Asp Val Pro Asp Phe Asn 35 40 45 actcaa att aca gag cca act ggg gag gga gac cgt ggg gat gtg gtc 193 Thr GlnIle Thr Glu Pro Thr Gly Glu Gly Asp Arg Gly Asp Val Val 50 55 60 gac gaaacc gat ttg tcc acg gat att gtc cca gag acc gag gct gct 241 Asp Glu ThrAsp Leu Ser Thr Asp Ile Val Pro Glu Thr Glu Ala Ala 65 70 75 80 tcc ttcgcc gct agt tca gta tct gca gcc tca cca gca tct gac acc 289 Ser Phe AlaAla Ser Ser Val Ser Ala Ala Ser Pro Ala Ser Asp Thr 85 90 95 gac agg cttctc tac tca acc tcc atg ccc gcc ttc ttg act gct aag 337 Asp Arg Leu LeuTyr Ser Thr Ser Met Pro Ala Phe Leu Thr Ala Lys 100 105 110 cgc aat aagaac ccc ggc aac ttg gac tgg agc gat gat gga tgc agc 385 Arg Asn Lys AsnPro Gly Asn Leu Asp Trp Ser Asp Asp Gly Cys Ser 115 120 125 aac tcc ccggac agg cct gca ggg ttt aac ttc ctt gac tc 426 Asn Ser Pro Asp Arg ProAla Gly Phe Asn Phe Leu Asp Ser 130 135 140 gtaagtcctc cttcatttatgctatctaca ttcactaata ttcgaacag c tgc aag 482 Cys Lys cgt cac gac ttcggg tac cgc aac tac aag aag cag cgc cgc ttc aca 530 Arg His Asp Phe GlyTyr Arg Asn Tyr Lys Lys Gln Arg Arg Phe Thr 145 150 155 160 gag cct aatcgc aag cgc att gat gac aat ttc aag aag gac cta tat 578 Glu Pro Asn ArgLys Arg Ile Asp Asp Asn Phe Lys Lys Asp Leu Tyr 165 170 175 aat gag tgcgcc aag tac tct ggc ctc caa tcc tgg aaa ggt gtt gcc 626 Asn Glu Cys AlaLys Tyr Ser Gly Leu Gln Ser Trp Lys Gly Val Ala 180 185 190 tgc cgc aaaatc gcg aac act tac tac gat gct gta cgc tcc ttc ggt 674 Cys Arg Lys IleAla Asn Thr Tyr Tyr Asp Ala Val Arg Ser Phe Gly 195 200 205 tgg ttgtaaatgtgcg gaagagatat caagtgggat cgaggaagag gatggtgaaa 730 Trp Leu 210gagctgagag gtggatttct ttacattccg caatggctac tacagaagaa ctgtgctcct 790caaatttaat ctcatttttg tgtctatcta tccactctag aa 832 10 210 PRT Tuberalbidum 10 Met Val Lys Ile Ala Ala Ile Val Leu Leu Met Gly Ile Leu AlaAsn 1 5 10 15 Ala Ala Ala Ile Pro Val Ser Glu Pro Ala Ala Leu Ala LysArg Gly 20 25 30 Asn Ala Glu Val Ile Ala Glu Gln Thr Gly Asp Val Pro AspPhe Asn 35 40 45 Thr Gln Ile Thr Glu Pro Thr Gly Glu Gly Asp Arg Gly AspVal Val 50 55 60 Asp Glu Thr Asp Leu Ser Thr Asp Ile Val Pro Glu Thr GluAla Ala 65 70 75 80 Ser Phe Ala Ala Ser Ser Val Ser Ala Ala Ser Pro AlaSer Asp Thr 85 90 95 Asp Arg Leu Leu Tyr Ser Thr Ser Met Pro Ala Phe LeuThr Ala Lys 100 105 110 Arg Asn Lys Asn Pro Gly Asn Leu Asp Trp Ser AspAsp Gly Cys Ser 115 120 125 Asn Ser Pro Asp Arg Pro Ala Gly Phe Asn PheLeu Asp Ser Cys Lys 130 135 140 Arg His Asp Phe Gly Tyr Arg Asn Tyr LysLys Gln Arg Arg Phe Thr 145 150 155 160 Glu Pro Asn Arg Lys Arg Ile AspAsp Asn Phe Lys Lys Asp Leu Tyr 165 170 175 Asn Glu Cys Ala Lys Tyr SerGly Leu Gln Ser Trp Lys Gly Val Ala 180 185 190 Cys Arg Lys Ile Ala AsnThr Tyr Tyr Asp Ala Val Arg Ser Phe Gly 195 200 205 Trp Leu 210 11 961DNA Verticillium tenerum CDS (5)..(628) 11 caac atg aag acc acc gct gttctc tcc ctc gcc atg ctc cag gcc acc 49 Met Lys Thr Thr Ala Val Leu SerLeu Ala Met Leu Gln Ala Thr 1 5 10 15 tgg gcc tcg ccc gtg gcc aag cgccag aac gac gtc tcc ctc gtc gac 97 Trp Ala Ser Pro Val Ala Lys Arg GlnAsn Asp Val Ser Leu Val Asp 20 25 30 aac tac atg ttc ggc atc tcg ctg cccacc ttc tcc aac cac cac tcc 145 Asn Tyr Met Phe Gly Ile Ser Leu Pro ThrPhe Ser Asn His His Ser 35 40 45 aac agg aac ccc cct cgc ctg gac tgg accacc gac ggc tgc acc tcg 193 Asn Arg Asn Pro Pro Arg Leu Asp Trp Thr ThrAsp Gly Cys Thr Ser 50 55 60 tcg ccc aac aac ccg ctc ggc ttc ccc ttc ctgccc gcc tgc cac cgc 241 Ser Pro Asn Asn Pro Leu Gly Phe Pro Phe Leu ProAla Cys His Arg 65 70 75 cac gac ttt ggc tac cag aac ttc cgc atc cag agccgc ttc acc cag 289 His Asp Phe Gly Tyr Gln Asn Phe Arg Ile Gln Ser ArgPhe Thr Gln 80 85 90 95 agc aac aag ctc cgc atc gac gac aag ttc aag gaggac ctc tac cac 337 Ser Asn Lys Leu Arg Ile Asp Asp Lys Phe Lys Glu AspLeu Tyr His 100 105 110 cag tgc gac ggc cac tgg gcc tgg gtt gcc tgc gctgcc ctc gcc gag 385 Gln Cys Asp Gly His Trp Ala Trp Val Ala Cys Ala AlaLeu Ala Glu 115 120 125 gtc tac tac gcc gcc gtc cgc gcc ttc ggc ggt ggtgac gcc acc ccg 433 Val Tyr Tyr Ala Ala Val Arg Ala Phe Gly Gly Gly AspAla Thr Pro 130 135 140 gga cgc atg cac gtc gcc gtc ttc ggc cag acc caggcc gag cac gac 481 Gly Arg Met His Val Ala Val Phe Gly Gln Thr Gln AlaGlu His Asp 145 150 155 gcc ctc gtc tcc atc tac gag gag aag ctc gcg gcctac gag gct gcc 529 Ala Leu Val Ser Ile Tyr Glu Glu Lys Leu Ala Ala TyrGlu Ala Ala 160 165 170 175 gtc gcc gag gcc gag gcc cgc ggc gag atc ccccac gtc gag gag acc 577 Val Ala Glu Ala Glu Ala Arg Gly Glu Ile Pro HisVal Glu Glu Thr 180 185 190 ctc ccc gag gag cct gcc gcc gag gag ccc gccgcc gag gag gag cag 625 Leu Pro Glu Glu Pro Ala Ala Glu Glu Pro Ala AlaGlu Glu Glu Gln 195 200 205 aag taaacacgag ccccttttag gaccgactagctcggtgtcg ctgggctagg 678 Lys ctgagctgag tgacggggag gcacgaaagagagcaatgca tcagacaggc tggaacatgc 738 ctttgtctga gtgatggatg gacttgatggacttgatgga cttggatgca tttatgatac 798 cgccagtgtt gactggcaga gcgagcgacttgattttgga tttcttgaaa ggacggatgt 858 cccgaggtgg ataagggatg ccttatcaccaacttcttca tgtatatatt gtactgcgca 918 gagaagcgcg ccccgaaaaa tggattgattcttgatgaga cgt 961 12 208 PRT Verticillium tenerum 12 Met Lys Thr ThrAla Val Leu Ser Leu Ala Met Leu Gln Ala Thr Trp 1 5 10 15 Ala Ser ProVal Ala Lys Arg Gln Asn Asp Val Ser Leu Val Asp Asn 20 25 30 Tyr Met PheGly Ile Ser Leu Pro Thr Phe Ser Asn His His Ser Asn 35 40 45 Arg Asn ProPro Arg Leu Asp Trp Thr Thr Asp Gly Cys Thr Ser Ser 50 55 60 Pro Asn AsnPro Leu Gly Phe Pro Phe Leu Pro Ala Cys His Arg His 65 70 75 80 Asp PheGly Tyr Gln Asn Phe Arg Ile Gln Ser Arg Phe Thr Gln Ser 85 90 95 Asn LysLeu Arg Ile Asp Asp Lys Phe Lys Glu Asp Leu Tyr His Gln 100 105 110 CysAsp Gly His Trp Ala Trp Val Ala Cys Ala Ala Leu Ala Glu Val 115 120 125Tyr Tyr Ala Ala Val Arg Ala Phe Gly Gly Gly Asp Ala Thr Pro Gly 130 135140 Arg Met His Val Ala Val Phe Gly Gln Thr Gln Ala Glu His Asp Ala 145150 155 160 Leu Val Ser Ile Tyr Glu Glu Lys Leu Ala Ala Tyr Glu Ala AlaVal 165 170 175 Ala Glu Ala Glu Ala Arg Gly Glu Ile Pro His Val Glu GluThr Leu 180 185 190 Pro Glu Glu Pro Ala Ala Glu Glu Pro Ala Ala Glu GluGlu Gln Lys 195 200 205 13 29 DNA Artificial TbPLA1 primer 13 caaggatccaaaatggtcaa gattgctgc 29 14 34 DNA Artificial TbPLA2 primer 14 tgcctcgagttttttctaga gtggatagat agac 34 15 690 DNA Fusarium venenatum CDS(49)..(597) 15 cagttttggt tctttccttc cttatccatc acttctagta tcttcaag atgaag ttc 57 Met Lys Phe 1 agc gct acc att ctt tca ctc ctc ccg gca gtt ctcgcc ctg ccc aca 105 Ser Ala Thr Ile Leu Ser Leu Leu Pro Ala Val Leu AlaLeu Pro Thr 5 10 15 ggc gaa gat gca tct gtc tca aag cgc cag agc gtg aacaca gtg aca 153 Gly Glu Asp Ala Ser Val Ser Lys Arg Gln Ser Val Asn ThrVal Thr 20 25 30 35 gat cag ctc ctc ttc agc gtc aca ctc cca caa ttc actgct cgt cgt 201 Asp Gln Leu Leu Phe Ser Val Thr Leu Pro Gln Phe Thr AlaArg Arg 40 45 50 aac gcc cgt gat cct ccc act gtc gac tgg acc tct gac ggttgc act 249 Asn Ala Arg Asp Pro Pro Thr Val Asp Trp Thr Ser Asp Gly CysThr 55 60 65 tcc tcg ccc gac aac cct ttc ggc ttc cct ttt atc cct gcc tgcaac 297 Ser Ser Pro Asp Asn Pro Phe Gly Phe Pro Phe Ile Pro Ala Cys Asn70 75 80 cgt cac gac ttt ggc tac cac aac tac cgc gcc cag agc cgc ttc acc345 Arg His Asp Phe Gly Tyr His Asn Tyr Arg Ala Gln Ser Arg Phe Thr 8590 95 gtg agc gcc aag tcc cgc atc gac aac aac ttc aag acc gat ttg tac393 Val Ser Ala Lys Ser Arg Ile Asp Asn Asn Phe Lys Thr Asp Leu Tyr 100105 110 115 ttc caa tgc caa tcc tcc agt gtt tct ggt gtc tgc aga gca cttgcc 441 Phe Gln Cys Gln Ser Ser Ser Val Ser Gly Val Cys Arg Ala Leu Ala120 125 130 gac gtc tac ttc gcc gcg gtt aga gct ttt ggc ggg gat gat gctact 489 Asp Val Tyr Phe Ala Ala Val Arg Ala Phe Gly Gly Asp Asp Ala Thr135 140 145 cct ggc aag aga gat gag gcc ctt gta aag gag tac gaa aag aaggta 537 Pro Gly Lys Arg Asp Glu Ala Leu Val Lys Glu Tyr Glu Lys Lys Val150 155 160 gaa gtc tac aac aag ctt gtt gaa gag gct cag aag aag ggt gatctc 585 Glu Val Tyr Asn Lys Leu Val Glu Glu Ala Gln Lys Lys Gly Asp Leu165 170 175 cct cgc ctt gac tagagtggtt caaaaagcat tctttgggtt cattgtacat637 Pro Arg Leu Asp 180 aaatccttac gatacatgag ttatgataaa tcttaaatggcgggtgacga gct 690 16 183 PRT Fusarium venenatum 16 Met Lys Phe Ser AlaThr Ile Leu Ser Leu Leu Pro Ala Val Leu Ala 1 5 10 15 Leu Pro Thr GlyGlu Asp Ala Ser Val Ser Lys Arg Gln Ser Val Asn 20 25 30 Thr Val Thr AspGln Leu Leu Phe Ser Val Thr Leu Pro Gln Phe Thr 35 40 45 Ala Arg Arg AsnAla Arg Asp Pro Pro Thr Val Asp Trp Thr Ser Asp 50 55 60 Gly Cys Thr SerSer Pro Asp Asn Pro Phe Gly Phe Pro Phe Ile Pro 65 70 75 80 Ala Cys AsnArg His Asp Phe Gly Tyr His Asn Tyr Arg Ala Gln Ser 85 90 95 Arg Phe ThrVal Ser Ala Lys Ser Arg Ile Asp Asn Asn Phe Lys Thr 100 105 110 Asp LeuTyr Phe Gln Cys Gln Ser Ser Ser Val Ser Gly Val Cys Arg 115 120 125 AlaLeu Ala Asp Val Tyr Phe Ala Ala Val Arg Ala Phe Gly Gly Asp 130 135 140Asp Ala Thr Pro Gly Lys Arg Asp Glu Ala Leu Val Lys Glu Tyr Glu 145 150155 160 Lys Lys Val Glu Val Tyr Asn Lys Leu Val Glu Glu Ala Gln Lys Lys165 170 175 Gly Asp Leu Pro Arg Leu Asp 180

1. A method of producing a phospholipase which comprises processing anexpressed fungal peptide so as to cleave off a peptide from theC-terminal end and/or a peptide from the N-terminal end to obtain a corepeptide with phospholipase activity, wherein the core peptide comprisesa) the amino acid sequence given by amino acids 146-153 of SEQ ID NO: 1,amino acids 87-94 of SEQ ID NO: 3, or amino acids 79-86 of SEQ ID NO:12; or a sequence identical to any of these amino acid sequences exceptfor the substitution of a single amino acid with another amino acid; andb) at least two cysteine residues located on the N-terminal side of thesequence given in a); and c) at least two cysteine residues located onthe C-terminal side of the sequence given in a).
 2. The method of claim1 wherein the expressed peptide is expressed in a filamentous fungalhost cell transformed with DNA encoding the expressed peptide.
 3. Themethod of claim 2 wherein the host cell is an Aspergillus, Fusarium orTrichoderma, particularly A. oryzae, A. niger, F. venenatum or T.reesei.
 4. The method of claim 2 wherein the expressed peptide isprocessed in vivo by the host cell.
 5. The method of claim 1 wherein thecore peptide has a length of 100-150 amino acids.
 6. The method of claim1 wherein the phospholipase has a specific phospholipase activity whichis at least 2 times the activity of the expressed peptide beforeprocessing.
 7. The method of claim 1 wherein the expressed peptide isderived from Tuber, Verticillium, Neurospora, Aspergillus, orHelicosporum, particularly T. borchii, T. albidum, V. dahliae, V.tenerum, N. crassa, A. oryzae, or Helicosporium sp.HN1.
 8. The method ofclaim 1 wherein the expressed peptide is cleaved within 0-18 amino acidson the N-terminal side of the sequence aligning with amino acids 97-101of SEQ ID NO: 1, when the complete expressed peptide sequence is alignedsimultaneously with the sequences given in SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10,and SEQ ID NO:
 12. 9. The method of claim 1 wherein the expressedpeptide is cleaved within 0-11 amino acids on the C-terminal side of thesequence aligning with amino acids 204-209 of SEQ ID NO: 1, when thecomplete expressed peptide sequence is aligned simultaneously with thesequences given in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO:
 12. 10. Themethod of any of claims 19 wherein the expressed peptide is cleavedwithin 10 amino acids of a Kex2 processing site or within 11 amino acidsof an FG sequence or both.
 11. A method of hydrolyzing a phospholipidcomprising contacting the phospholipid with a phospholipase produced bythe method of any of the claims
 140. 12. A method of producing cheesecomprising contacting cheese milk or a fraction of cheese milk with aphospholipase, wherein the phospholipase comprises: d) the sequencegiven by amino acids 146-153 of SEQ ID NO: 1, amino acids 87-94 of SEQID NO: 3, or amino acids 79-86 of SEQ ID NO: 12; or a sequence identicalto any of these amino acid sequences except for the substitution of asingle amino acid with another amino acid; and e) two cysteine residueslocated on the N-terminal side of the sequence given in a); and f) twocysteine residues located on the C-terminal side of the sequence givenin a).
 13. A method of producing cheese comprising contacting cheesemilk or a fraction of cheese milk with a phospholipase produced by themethod of claim
 1. 14. A phospholipase which is a polypeptide having anamino acid sequence which is at least 80% identical with amino acids91-210 in SEQ ID NO: 10 (T. albidum), amino acids 92-211 in SEQ ID NO: 1(T. borchii), amino acids 30-137 in SEQ ID NO: 12 (V. tenerum), aminoacids 38-145 in SEQ ID NO: 3 (V. dahliae), amino acids 44-151 in SEQ IDNO: 4 (N. crassa), amino acids 37-157 in SEQ ID NO: 7 (A. oryzae), oramino acids 58-168 in SEQ ID NO: 8 (N. crassa).
 15. A phospholipasewhich comprises: g) a polypeptide encoded by the phospholipase encodingpart of the DNA sequence cloned into a plasmid present in Escherichiacoli deposit number DSM 15442; or h) a polypeptide comprising the aminoacid sequence of amino acids 29 to 149 of SEQ ID NO: 16, or an aminoacid sequence which can be obtained therefrom by substitution, deletion,and/or insertion of one or more amino acids; or i) an analogue of thepolypeptide defined in (a) or (b) which: i) has at least 80% homologywith said polypeptide, or ii) is immunologically reactive with anantibody raised against said polypeptide in purified form, or iii) is anallelic variant of said polypeptide; or j) a polypeptide which isencoded by a nucleic acid sequence which hybridizes under low stringencyconditions with a complementary strand of the nucleic acid sequence ofnucleic acids 133 to 495 SEQ ID NO: 15 encoding the mature polypeptideor a subsequence thereof having at least 100 nucleotides.
 16. Thephospholipase of claim 15 which is native to a strain of Fusarium,particularly F. venenatum.
 17. A nucleic acid sequence comprising anucleic acid sequence which encodes the phospholipase of claim
 15. 18. Anucleic acid sequence which comprises: k) the partial DNA sequenceencoding a mature phospholipase cloned into a plasmid present inEscherichia coli DSM 15442, l) the partial DNA sequence encoding amature phospholipase of nucleic acids 133 to 495 of SEQ ID NO: 15, m) ananalogue of the sequence defined in a) or b) which encodes aphospholipase and i) has at least 80% homology with said DNA sequence,or ii) hybridizes at high stringency with a complementary strand of saidDNA sequence or a subsequence thereof having at least 100 nucleotides,iii) is an allelic variant thereof, or n) a complementary strand of a),b) or c).
 19. A nucleic acid construct comprising the nucleic acidsequence of claims 17 operably linked to one or more control sequencescapable of directing the expression of the phospholipase in a suitableexpression host.
 20. A recombinant expression vector comprising thenucleic acid construct of claim 19, a promoter, and transcriptional andtranslational stop signals. 21-28 (Cancelled)